Low molecular weight heparins (LMWHs) are partially depolymerized natural products of heparin, which are isolated from porcine intestine. A worldwide distribution of contaminated heparin in 2007 was associated with 85 deaths in the US. This crisis revealed the vulnerability of the LMWH supply chain. LMWHs are complex mixtures, having average molecular masses of 3500-6000 Daltons, corresponding to 12-20 saccharide units. The efficient preparation of a synthetic LMWH could improve the safety, availability and efficacy. However, the production of homogeneous LMWHs has not been possible due to difficulties in the chemical synthesis. Glycan Therapeutics proposes to assess an innovative chemoenzymatic approach that would provide an unprecedented efficiency in the preparation of heparin. Our product will be animal- free, structurally homogenous, safe for renal-impaired patients, and have the ability of its anticoagulant activity to be neutralized by protamine. In the phase I STTR studies, we have identitifed a heparin dodecasaccharide (12-mer) as a candidate to substitute animal-sourced LMWH. We also demonstrated the ability to synthesize this 12-mer at the scale of 20 g in four batches. In the presently proposed Phase II SBIR studies, we will assess the feasibility of a larger scale synthesis (200 grams) to conduct IND-enabling studies. Aim 1 will develop a process to prepare 50 g of the 12-mer via a series of efforts for improving enzyme production, reaction efficacy and simplifying purifications. Aim 2 will examine the immunogenicity of the 12- mer in a murine model. We will also conduct 14-day toxicity studies in rat and dog as well as related IND-enabling studies. The completion of this project will modernize this century-old drug with improved safety and anticoagulant properties.